The present invention relates to the field of plant biotechnology. More specifically, it concerns methods of incorporating genetic components into a plant comprising a T-DNA transfer process. In particular, provided herein are systems for genetically transforming monocotyledonous plants including corn, rice, and wheat.
The method comprises novel conditions during the inoculation, co-culture, or infiltration of Agrobacterium with a transformable plant cell or tissue. Exemplary methods include an improved method using a bacterial growth suppressing agent during the Agrobacterium-mediated transformation process. The improved method can be used for introducing nucleic acids into transformable cells or tissues using a variety of selectable and/or screenable marker systems, and with a number of different plant species. The present invention also provides transgenic plants, in particular, corn, rice, and wheat. In other aspects, the invention relates to the production of stably transformed plants, gametes, and offspring from these plants.
During the past decade, it has become possible to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology. This advance has provided enormous opportunities to improve plant resistance to pests, disease and herbicides, and to modify biosynthetic processes to change the quality of plant products (Knutson et al., 1992; Piorer et al., 1992). However, the availability of efficient Agrobacterium-mediated transformation methods suitable for high capacity production of economically important plants is limited. In particular, a novel culture system that generates reproducible transformants with a simple integration pattern of the introduced DNA into the host genome, more specifically, the integration of a low copy number (one to two copies) of the introduced DNA is needed.
There have been many methods attempted for plant transformation, but only a few methods are highly efficient. Moreover, few methods are both highly efficient and result in transformants with simple integration pattern and low copy number of the introduced DNA. Copy number refers to the number of complete or incomplete copies of T-DNA introduced in host cell. The technologies for the introduction of DNA into cells are well known to those of skill in the art and can be divided into categories including but not limited to: (1) chemical methods (Graham and van der Eb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation ( Fromm et al., 1985; U.S. Pat. No. 5,384,253) and the gene gun (Christou, 1992; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988);(4) receptor-mediated mechanisms (Curiel et al., 1992); and (5) Agrobacterium-mediated plant transformation methods.
Until recently, the methods employed for some monocot species included direct DNA transfer into isolated protoplasts and microprojectile-mediated DNA delivery (Fromm et al, 1990). The protoplast methods have been widely used in rice, where DNA is delivered to the protoplasts through liposomes, PEG, and electroporation. While a large number of transgenic plants have been recovered in several laboratories (Datta et al., 1990), the protoplast methods require the establishment of long-term embryogenic suspension cultures. Some regenerants from protoplasts are infertile and phenotypically abnormal due to the long-term suspension culture (Davey et al., 1991; Rhodes et.al.,1988). U.S. Pat. No. 5,631,152 describes a rapid and efficient microprojectile bombardment method for the transformation and regeneration of monocots and dicots.
To date, microparticle- and Agrobacterium-mediated gene delivery are the two most commonly used plant transformation methods. Microparticle-mediated transformation refers to the delivery of DNA coated onto microparticles that are propelled into target tissues by several methods. This method can result in transgenic events with a higher copy number, complex integration patterns, and fragmented inserts. Agrobactenum-mediated plant transformation can also result in transformed plants with multiple copies of inserts and complex integration patterns. A reduction in copy number can result from a decrease in the frequency of T-DNA transfer. Accordingly, novel culture conditions can be manipulated to impact the frequency of T-DNA transfer and can produce transformation events containing the optimum number of copies of the introduced DNA.
A reproducible Agrobacterium-mediated method that consistently results in low copy number inserts and is applicable to a broad number of plant species is desirable for a number of reasons. For example, the presence of multiple inserts can lead to a phenomenon known as gene silencing which can occur by several mechanisms including but not limited to recombination between the multiple copies which can lead to subsequent gene loss. Also, multiple copies can cause reduced levels of expression of the gene which in turn can result in the reduction of the characteristic(s) conferred by the gene product(s). Despite the number of transformation methods available for specific plant systems, it would be advantageous to have a method of introducing genes into plants that is applicable to various crops and a variety of transformable tissues.
Agrobacterium-mediated transformation is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrobacterium. Several Agrobacterium species mediate the transfer of a specific DNA known as xe2x80x9cT-DNAxe2x80x9d, that can be genetically engineered to carry any desired piece of DNA into many plant species. The major events marking the process of T-DNA mediated pathogenesis are: induction of virulence genes, processing and transfer of T-DNA, This process is the subject of many reviews (Ream, 1989; Howard and Citovsky, 1990; Kado, 1991; Hooykaas and Schilperoort, 1992; Winnans, 1992; Zambryski, 1992; Gelvin, 1993; Binns and Howitz, 1994; Hooykaas and Beijersbergen 1994; Lessl and Lanka, 1994; Zupan and Zambryski, 1995).
Agrobacterium-mediated genetic transformation of plants involves several steps. The first step, in which the Agrobacterium and plant cells are first brought into contact with each other, is generally called xe2x80x9cinoculationxe2x80x9d. Following the inoculation step, the Agrobacterium and plant cells/tissues are usually grown together for a period of several hours to several days or more under conditions suitable for growth and T-DNA transfer. This step is termed xe2x80x9cco-culturexe2x80x9d. Following co-culture and T-DNA delivery, the plant cells are often treated with bacteriocidal and-or bacteriostatic agents to kill the Agrobacterium. If this is done in the absence of any selective agents to promote preferential growth of transgenic versus non-transgenic plant cells, then this is typically referred to as the xe2x80x9cdelayxe2x80x9d step. If done in the presence of selective pressure favoring tranasgenic plant cells, then it is referred to as a xe2x80x9cselectionxe2x80x9d step. When a xe2x80x9cdelayxe2x80x9d is used, it is followed by one or more xe2x80x9cselectionxe2x80x9d steps. Both the xe2x80x9cdelayxe2x80x9d and xe2x80x9cselectionxe2x80x9d. steps typically include bacteriocidal and-or bacteriostatic agents to kill any remaining Agrobacterium cells because the growth of Agrobacterium cells is undesirable after the infection (inoculation and co-culture) process.
Although transgenic plants produced through Agrobacterium-mediated transformation generally contain a simple integration pattern as compared to microparticle-mediated genetic transformation, a wide variation in copy number and insertion patterns exists (Jones et al, 1987; Jorgensen et al., 1987). Moreover, even within a single plant genotype, different patterns of T-DNA integration are possible based on the type of explant and transformation system used (Grevelding et al., 1993). Factors that regulate T-DNA copy number are poorly understood. A reproducible, broadly applicable method to increase the efficiency of producing plants with a low copy number, and preferably a single copy of the T-DNA would be highly desirable to those practicing in the art.
Recently, monocot species have been successfully transformed via Agrobacterium-mediated transformation. WO 97/48814 discloses processes for producing stably transformed fertile wheat. The method describes the recovery of transgenic, wheat plants within a short period of time using a variety of explants. Agrobacterium-mediated transformation provides a viable alternative to bombardment methods and the method also allows more efficient molecular characterization of transgenic lines. The present invention is an improved Agrobacterium-mediated transformation method that relies on the control of Agrobacterium growth during the transformation process. More specifically, the present invention focuses on controlling Agrobacterium growth in the stages of Agrobacterium-mediated transformation during which T-DNA transfer can occur.
The major deficiencies in current plant transformation systems utilizing Agrobacterium-mediated methods include but are not limited to the production efficiency of the system, and transformation difficulties due to genotype or species diversity and explant limitations. WO 94/00977 describes a method for transforming monocots that depends on the.use of freshly cultured immature embryos for one monocot and cultured immature embryos or callus for a different monocot. In either system, the explants must be freshly isolated, and the method is labor intensive, genotype-, and explant-limited. Other reports rely on the use of specific strains or vectors to achieve high efficiency transformation. In one report, a specific super-binary vector must be used in order to achieve high-efficiency transformation (Ishida et al., 1996).
Despite the number of transformation methods in the art, few methods have been developed that are broadly applicable to genotypes of a single crop species as well as to genotypes of other crop species. What is lacking in the art is an Agrobacterium-mediated plant transformation system that is efficient, reproducible, applicable to a number of plant systems, and a transformation system that effectively results in transformed plants with a simple integration pattern and a low copy number. The present invention provides novel culture conditions using one or more bacterial growth inhibiting agents during inoculation and co-culture of Agrobacterium with a transformable plant cell or tissue that result in increased transformation efficiencies and a low copy number of the introduced genetic component in several plant systems. The method of the present invention consistently results in desired transgenic events with a low number of inserts and reduces the need to screen hundreds of lines for identification of the optimal commercial line for breeding and introduction of improved germplasm to plant breeders, growers, and consumers. The present invention thus provides a novel improvement compared to existing Agrobacterium-mediated transformation methods.
The present invention provides novel methods for the stable and efficient transformation of plants under conditions that inhibit the growth of Agrobacterium cells during the transformation process.
In one aspect the present invention provides a novel method of transforming a plant cell or plant tissue with Agrobacterium by inoculating a transformabe cell or tissue containing at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent, co-culturing in the presence or absence of the growth inhibiting agent, selecting a transformed plant cell or tissue, and regenerating a transformed plant expressing the genetic component from the selected plant cells or tissues.
In one embodiment, the growth inhibiting agent comprises a compound containing a heavy metal such as silver, or an antibiotic such as carbenicillin, or a nucleic acid, or protein capable of inhibiting or suppressing the growth of Agrobacterium cells and the growth inhibiting agent is present during the inoculation step in the transformation process and not in the co-culture step.
In another embodiment, the growth inhibiting agent that is inhibitory to Agrobacterium cell growth is absent during the inoculation step, but present in the co-culture step in the transformation process.
In still another embodiment the invention relates to the presence of at least one Agrobacterium growth inhibiting agent during the inoculation process in an amount sufficient to suppress Agrobacterium growth and reduce T-DNA transfer, thus favoring low copy insertions of the introduced DNA.
Still another aspect of the present invention relates to transformed plants produced by inoculating a transformable cell or tissue containing at least of at least one genetic component capable of being transferred to the plant cell or tissue in the presence of at least one growth inhibiting agent, co-culturing in the presence or absence of the growth inhibiting agent, selecting a transformed plant cell or tissue and regenerating a transformed plant expressing the genetic component from the selected plant cells or tissues.
Yet another aspect of the present invention relates to any seeds, or progeny of the transformed plants produced by the method of the present invention.
Further objects, advantages and aspects of the present invention will become apparent from the accompanying figures and description of the invention.